Cryptosporidium parvum

Cryptosporidium parvum is a coccidian protozoan that causes diarrhoea in human, usually self-limited in immunocompetent hosts but severe and debilitating in immunocompromised hosts (Guerrent 1997). Cryptosporidiosis is very common in patients with AIDS (Balatbat et al. 1996). Commonly utilized coprodiagnostic methods involving stool concentration and vizualization using acid-fast (AF) or immunofluorescent staining (IF) have a low sensitivity, especially in formed stools, in which the threshold for detection may require 50000 (IF) to 500000 (AF) oocysts per gram of stool (Weber et al.

1991). Development of more sensitive methods would, therefore, have significant clinical implications. Laxer et al. (1991) reported a C. parvum specific PCR, which was evaluated on human stool samples and on fixed, paraffin-embedded tissue samples (Laxer et al. 1992). The assay was capable to detect only one parasite. The same primer sequences were used by Gobet et al. (1997), who introduced hypochlorite inactivation of the samples. A more simplified oocyst preparation procedure, combined with a nested PCR, detected 500 oocysts per gram of stool or 500ng C. parvum DNA (Balatbat et al. 1996). Diagnostic primers for Cryptosporidium spp.

have also been developed utilizing the random amplified polymorphic DNA (RAPD) analysis, which also have been used to genetically differentiate isolates of Cryptosporidium spp. (Morgan et al. 1995). In addition, the 18S sequence of rRNA genes of C. parvum and C. muris have been used in an assay, based on PCR and endonuclease restriction, for detection and species identification (Awad-el-Kariem et al. 1994). The increased sensitivity of the PCR assays, compared to current methods, may also contribute to identification of carriers of C. parvum, with or without active diseases, at a threshold below what is detectable by the current methods (Balatbat et al. 1996).

Another application of Cryptosporidia-PCR is to investigate the presence of Cryptosporidia-species in environmental water (surface, drinking water, and sewage samples) (Johnson et al. 1995; Stinear et al. 1996; Rochelle et al. 1997).

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